Siniperca chuatsi il-6 gene and detection method of disease-resistant snp marker thereof

ABSTRACT

The invention provides a  Siniperca chuatsi  IL-6 gene and a detection method for a disease-resistant SNP marker. A cDNA sequence of  S. chuatsi  IL-6 gene is cloned, as shown in SEQ ID NO: 1. A IL-6 gene gDNA sequence containing an intron of the  S. chuatsi  IL-6 gene is cloned, as shown in SEQ ID NO: 2. A primer for amplifying a disease-resistant SNP locus is designed according to IL-6 gDNA sequence, and  S. chuatsi  IL-6 gene is amplified to obtain an amplification product which is sequenced, and the SNPs loci relevant to virus disease-resistance are found out and the SNP locus is determined according to DNA peak profile. The IL-6 cDNA full-length sequence and IL-6 gDNA full-length sequence are cloned firstly. The SNP locus relevant to virus disease resistance of  S. chuatsi  IL-6 gene is detected, thereby providing a new method for breeding of  S. chuatsi.

This application is a Divisional Application of U.S. Ser. No.16/504,371, filed on Jul. 8, 2019, which claims priority to ChinesePatent Application No.: 201810734995.2, filed on Jul. 6, 2018, which isincorporated by reference for all purposes as if fully set forth herein.

FIELD OF THE INVENTION

The present invention relates to the fields of aquiculture andbiotechnology, and more particularly to Siniperca chuatsi (S. chuatsi)IL-6 gene and a detection method of a disease-resistant SNP markerthereof.

DESCRIPTION OF THE RELATED ART

As a “prestigious” fish with important economic value in China, S.chuatsi occupies an important position in freshwater aquaculture inChina. However, under the conditions of large-scale artificial breedingin recent years, the genetical characterization decline of S. chuatsi isquite common, which leads to the gradual decline of disease resistance,especially the increasingly rampant viral diseases infected byinfectious spleen and kidney necrosis virus. And so far, there is noeffective prevention method, which causes serious economic losses everyyear and seriously restricts the sustainable development of theaquaculture industry.

The existing fish breeding technique is to select fish based on theapparent traits of fish observed by naked eyes, which makes itimpossible to correctly select fish from the perspective of geneticdifferences closely related to apparent traits. It is not onlyimpossible to carry out early breeding, but also difficult to guaranteethe offspring characteristics and the breeding is blind, time-consumingand laborious.

Cloning of genes related to disease resistance and screening ofdisease-resistant germplasm resources by SNP molecular markers areeffective means to improve disease resistance of S. chuatsi to virusesand other diseases. SNPs, that is, signal nucleotide poly-morphisms.transition, transversion, insertion or deletion of a single base on agene fragment will lead to SNP polymorphism and disease resistancephenotype differences among individuals.

During the disease-resistant genes, IL-6 (interleukin-6) is a cytokinebelonging to interleukin, which can promote the proliferation anddifferentiation of various cells involved in the immune responses,improve their functions, and inhibit cell apoptosis. However, currentlythe researches on the cloning and functions of fish IL-6 gene are lack,because the fish has low homology of IL-6 gene sequence with the otherspecies, moreover, the transcription and translation process of IL-6gene is very unstable when subjected to immunostimulation, and thuscauses fewer clones of cDNA library of fish cytokines. Accordingly,cloning of cDNA full-length sequence, gDNA full-length sequence of S.chuatsi IL-6 gene is of great significance.

SUMMARY OF THE INVENTION

An object of the invention is to provide a disease-resistant IL-6 gene(interleukin, IL-6) of S. chuatsi, intended to solve the problem thatthe fish has low IL-6 gene homology with other species and some fishlack of IL-6 gene.

Another object of the invention is to provide a detection method of adisease-resistant SNP marker of S. chuatsi. The method is based on theIL-6 gene of S. chuatsi provided in the invention, wherein singlenucleotide polymorphism (SNP) is combined with disease-resistantapparent traits of fish, based on SNP marker (or known as mutation site)of IL-6 of disease-resistant or non-disease-resistant fish, to breedselectively and accurately disease-resistant fish population. Theinvention can improve the efficiency of breeding of superior breeding,decrease the morbidity and mortality of the breeding fish, and thusincrease the economic benefits of fish culture, thereby providing asignificant meaning for the progress of fish breeding.

The invention utilizes the following technical solutions:

In one aspect, the invention provides a S. chuatsi IL-6 gene having acDNA sequence shown in SEQ ID NO: 1.

In another aspect, the invention also provides a detection method for adisease-resistant SNP marker based on the above IL-6 gene of S. chuatsi,comprising the steps of

(1) designing a primer for amplifying a disease-resistant SNP locusaccording to IL-6 gDNA sequence containing intron of the S. chuatsi IL-6gene, and amplifying the S. chuatsi IL-6 gene to obtain an amplificationproduct;

(2) sequencing the amplification product and finding out SNPs locirelevant to virus disease resistance, and determining the SNP locusaccording to DNA peak profile.

Preferably, in the step (1), the IL-6 gDNA sequence is obtained bydesigning and amplifying a specific primer for S. chuatsi IL-6 gene, andthe IL-6 gDNA sequence has a nucleotide sequence shown in SEQ ID NO: 2.

Preferably, the specific primer is:

(SEQ ID NO: 12) IL-6-1F: 5′-CTCAGCATCAGCGGAAACTC-3′; (SEQ ID NO: 13)IL-6-1R: 5′-TGCCCCTGTTGGCCATACTT-3′;

Preferably, in the step (1), rapid amplification of cDNA ends (RACE) isperformed to clone the S. chuatsi IL-6 gene.

Preferably, in the step (1), the primer for amplifying disease-resistantSNP loci is:

(SEQ ID NO: 14) IL-6- L1F: 5′-AACCCAAAGAGGCAGGTGAC-3′; (SEQ ID NO: 15)IL-6- L1R: 5′-ACCATCCAATTTCCCTGAGGT-3′.

Preferably, in the step (2), multiple sequence alignment is performed onthe sequencing results by DNAMAN software and the suspected SNPs lociare found out, and DNA sequencing chromatogram is observed via Chromassoftware, single peak is a homozygotic SNP locus, and the nested peak isa heterozygous SNP locus.

Preferably, the SNP marker is located at the 1744 bp of the gDNAsequence S. chuatsi having a nucleotide sequence shown in SEQ ID NO: 2,the base at 1744 bp of S. chuatsi susceptible to virus is T, and thebase at 1744 bp of antivirus S. chuatsi is C, the virus is infectiousspleen and kidney necrosis virus.

As compared with prior art, the present invention has the followingadvantages:

(1) In the invention, IL-6 cDNA full-length sequence and IL-6 gDNAfull-length sequence of S. chuatsi are cloned firstly, thereby providingresearch basis for resistant breeding of fish;

(2) In the invention, the SNP locus of mandarin fish IL-6 gene relevantto virus disease resistance is detected to provide a new idea forbreeding of S. chuatsi, this facilitates to promote the genetic breedingprocess and increase the economic benefits of mandarin fish culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the gel electrophoresis results of segments of S. chuatsiIL-6 gene after amplification, wherein (A) agarose gel electrophoresisresults of second round PCR of S. chuatsi IL-6 gene via cDNA 5′ endRACE, lane 1 represents the target fragment; (B) agarose gelelectrophoresis results of second round PCR of S. chuatsi IL-6 gene viacDNA 3′ end RACE, lane 1 represents the target fragment;

FIG. 2 shows the gel electrophoresis results after PCR amplification ofS. chuatsi IL-6 gene by adding the primer used by an intron;

FIG. 3 shows the gel electrophoresis results after PCR amplification ofSNP locus detection of S. chuatsi IL-6 gene;

FIG. 4 shows the PCR detection results of infectious spleen and kidneynecrosis virus (ISKNV) for head kidney tissue of S. chuatsi, whereinlane {circle around (1)} represents the band after DL500 Markelectrophoresis; both lane {circle around (2)} and lane {circle around(3)} represent a single and bright ISKNV virus nucleotide-specific band(187 bp) obtained by performing PCR amplification and electrophoresisusing a template prepared by head kidney tissue homogenate of S. chuatsiin the ISKNV virus infection group; the other lanes representnon-specific bands obtained by performing PCR amplification andelectrophoresis using a template prepared by head kidney tissuehomogenate of S. chuatsi in the ISKNV virus infection group ;

FIG. 5 shows a screen shot of comparison results of IL-6 DNA reversecomplementary sequence of 22 groups of S. chuatsi samples, wherein thebase G is mutated to base A in three samples.

FIG. 6 shows the peak profile generated by sequencing IL-6 DNA of a S.chuatsi sample, wherein G homozygote is not mutated in IL-6 DNA of S.chuatsi sample of this figure;

FIG. 7 shows the peak profile generated by sequencing IL-6 DNA of a S.chuatsi sample, wherein G heterozygote is not mutated in IL-6 DNA of aS. chuatsi sample of this figure.

FIG. 8 shows the peak profile generated by sequencing IL-6 DNA of a S.chuatsi sample, wherein A homozygote is mutated in IL-6 DNA of S.chuatsi sample of this figure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be explained in more detail below withreference to the drawings and in connection with embodiments. It shouldbe understood that, the exemplary embodiments and description thereof ofthe invention are used for illustrating the present invention and arenot intended to limit the invention

I Clone of IL-6 cDNA Sequence of S. chuatsi1. Amplification of IL-6 5′ Terminal Sequence of S. chuatsi

(1) Synthesis, Purification and Tailing of the First Strand of cDNA

The first strand of cDNA of IL-6 was synthesized for the extracted totalRNA using SUPERSCRIPT II RT enzyme and the primer GSP-IL-6-1. RNase Mixwas used to perform RNA removal on the synthesized cDNA. Reversetranscription system was:

GSP-IL-6-1 25 ng Template RNA 2 ul DEPC-treated water making up to 15.5μl

This system was incubated at 70° C. for 10 min, and immediately placedon ice to cool for 1 min, and then transient centrifugation wasperformed to collect liquid. The following system was addedsequentially:

10 × PCR bbuffer 2.5 μl 25 nM MgCl₂ 2.5 μl 10 nM dNTP mix   1 ul 0.1MDTT 2.5 ul

The reaction system was mixed gently, centrifuged and incubated at 42°C. for 1 min. 1 μl SUPERSCRIPT II RT was added, mixed evenly andincubated at 42° C. for 50 min, at 70° C. for 15 min and then reactionwas stopped. After centrifugation, the resulting product was placed at37° C., and 1 μl of RNase mix was added to react for 30 min and thenpurification was performed. poly (c) was added at the purified cDNAterminal using TdT enzyme and dCTP, then the product was stored at a lowtemperate for use.

(2) Quick Amplification of cDNA 5′ Ends

The first round PCR amplification was performed on the cDNA to which thedC tail had been added using the primer GSP-IL-6-2. The 5′-RACE systemwas added in the following order:

sterilized, distilled water 31.5 μl 10 × PCR buffer  5.0 μl 25 mM MgCl₂ 3.0 μl 10 mM dNTP mix  1.0 μl 10 μM nested GSP-TLR3-2  2.0 μl 10 uMAbridged Anchor Primer  2.0 μl dC-tailed cDNA  5.0 μl Taq DNA polymerase 0.5 μl final volume   50 μl

The reaction conditions were as follows: pre-denaturation at 94° C. for2 min, denaturation at 94° C. for 30 s, re-analysis annealing at 55° C.for 30 s, extension at 72° C. for 1 min, 30 cycles, and finallyextension at 72° C. for 7 min.

The nested second round PCR amplification was performed using the primerGSP-IL-6-3 and abridged universal amplification primer AUAP, and thesystem was as follows:

sterilized, distilled water 33.5 μl 10 × PCR buffer  5.0 μl 25 mM MgCl₂ 3.0 μl 10 mM dNTP mix  1.0 μl 10 μM nested GSP-TLR3-3  1.0 μl 10 uMAUAP  1.0 μl dilution of primary PCR product  5.0 μl Taq DNA polymerase 0.5 μl final volume   50 μl

The reaction conditions were as follows: pre-denaturation at 94° C. for2 min, denaturation at 94° C. for 30 s, re-analysis annealing at 59° C.for 30 s, extension at 72° C. for 1 min, 30 cycles, and finallyextension at 72° C. for 7 min.

Agarose gel electrophoresis (1.2%) was performed on the product of thesecond round PCR, the target bands were recycled by gel extraction usingGel Extraction Kit (Sangon Shanghai). The purified PCR product wasrecycled and cloned to the pMD18-T vector (TaKaRa), and positive cloneswere selected for sequencing.

2. Amplification of IL-6 3′ Terminal Sequence of S. chuatsi

(1) Synthesis and Purification of the First Strand of cDNA

Reverse transcription was performed on the total extracted RNA usingSUPERSCRIPT II RT enzyme and the primer 3′CDS primer A (SMARTer™ RACEcDNA Amplification Kit, Clontech). The used primer was 3′CDS primer A,and the other components and conditions were the same as those of 5′terminal amplification.

(2) Quick Amplification of cDNA Ends

The first round PCR amplification was performed using the primersGSP-IL-6-4 and UPM using the cDNA synthesized previously as a template,and the 3′-RACE reaction system was added in the following order (thereaction conditions were the same as those of the 5′ endsamplification):

sterilized, distilled water 31.5 μl 10 × PCR buffer  5.0 μl 25 mM MgCl₂ 3.0 μl 10 mM dNTP mix  1.0 μl 10 μM GSP-IL-6-4  2.0 μl 10 uM UPM  2.0μl dC-tailed cDNA  5.0 μl Taq DNA polymerase  0.5 μl final volume   50μl

The PCR product from the first round amplification was diluted 50 timesto perform the second round PCR amplification, the other systems werethe same as those of the first round PCR amplification other than theprimers GSP-IL-6-5 and UPM, and the reaction conditions were the same asthose of 5′ amplification. Agarose gel electrophoresis (1.2%) wasperformed on the product of the second round PCR, and the target bandwas recycled by gel extraction, as shown in FIG. 1. The purified productwas cloned to the pMD18-T vector (TaKaRa), and the positive clones wereselected for sequencing, the spliced complete sequence of S. chuatsiIL-6 cDNA is shown in SEQ ID NO: 1, and the encoded amino acid sequenceof the S. chuatsi IL-6 cDNA is shown in SEQ ID NO: 3.

TABLE 1 Primer Sequences for amplification of IL-6 gene cDNA PrimersSequences ( 5′-3′ ) Target GSP- IL-6-1 CTGGGGCACTCCTTCT IL-6 5′ -(SEQ ID NO: 4) Race GSP- IL-6-2 AACCTGTGGAGACAAGCC IL-6 5′ -(SEQ ID NO: 5) Race GSP- IL-6-3 CTGAAGTTGGAGTAAGGGCA IL-6 5′ -(SEQ ID NO: 6) Race 3′CDS Primer A AAGCAGTGGTATCAACGCAGACTA IL-6 3′-(SEQ ID NO: 7) C Race GSP- IL-6-4 CGCCAGCTCCACTACTTCCTTGTCG IL-6 3′-(SEQ ID NO: 8) Race GSP- IL-6-5 AAAGGGAGTTCAGAGCAAGTATGG IL-6 3′-(SEQ ID NO: 9) C Race AUAP GGCCACGCGTCGACTAGTAC universal(SEQ ID NO: 10) 5′ -Race UPM CTAATACGACTCACTATAGGGC universal(SEQ ID NO: 11) 3′-Race3. Obtaining of S. chuatsi IL-6 gDNA

With reference to the known IL-6 gDNA structure of other fishes, thespecific primers were designed for the spliced complete fragment of S.chuatsi IL-6 cDNA to amplify the intron of IL-6 gene (table 2), whereinthe fragment should contain overlapping regions for facilitating thesubsequent sequence assembly. The genomic DNA of S. chuatsi was used asa template to perform fragment PCR amplification. The amplificationconditions were as follows: pre-denaturation at 94° C. for 5 min,denaturation at 94° C. for 30 s, annealing at 52° C. for 30 s, extensionat 72° C. for 1 min, 30 cycles, and finally extension at 72° C. for 7min. Agarose gel electrophoresis (1.5%) was performed on the PCRproduct, as shown in FIG. 2, single band was selected and recycled bygel extraction, and ligated to the pUmc-T vector using T vector PCRproducts cloning Kit(TAKARA), the system(10 μL) was stored overnight at4° C. The positive clones were selected for sequencing. The obtainedsequences were spliced using artificial alignment and DNAMAN software toget the full-length sequence of S. chuatsi IL-6 gDNA, as shown in SEQ IDNO: 2.

TABLE 2 Primer sequences for amplifying the intron of IL-6 gene PrimersPrimer sequences (5′-3′) IL-6-1F (SEQ ID NO: 12) CTCAGCATCAGCGGAAACTCIL-6-1R (SEQ ID NO: 13) TGCCCCTGTTGGCCATACTT

4. Detection of Disease-Resistant SNP Marker

(1) Primers (Table 3) Were Designed According to the Sequence of S.chuatsi IL-6 gDNA. The PCR Reaction System is Shown in Table 4.

TABLE 3 Primer sequences for obtaining disease-resistantSNP loci by amplifying S. chuatsi IL-6 gene PrimersPrimer sequences ( 5′-3′ ) IL-6- L1F (SEQ ID NO: AACCCAAAGAGGCAGGTGAC14) IL-6- L1R (SEQ ID NO: ACCATCCAATTTCCCTGAGGT 15)

TABLE 4 PCR reaction system for obtaining disease-resistant SNP loci byamplifying S. chuatsi IL-6 gene Components Volume μl) Template DNA 2Primer R (10 μl) 1 Primer F (10 μl) 1 2 × Easy Taq PCR Super Mix 25ddH₂O 21 Total 50

Steps of PCR amplification were as follows: (1) pre-denaturation at 94°C. for 5 min, (2) denaturation at 94° C. for 30 s, (3) annealing at 52°C. for 30 s, (4) extension at 72° C. for 1 min, 30 cycles, and (5)extension at 72° C. for 10 min, (1) 4° C. end of the reaction.

(2) Agarose Gel Electrophoresis (1.5%) Was Performed on the PCR Productto Get the PCR Amplification Product, As Shown in FIG. 3, and ThenImmediately Sequenced After Gel Extraction.

The multiple sequence alignment was performed on the sequencing resultsusing DNAMAN software, to find out the SNPs loci relevant to the virusdisease resistance. Then DNA sequencing chromatogram was observed viaChromas software, single peak was a homozygotic SNP locus, and thenested peak was a heterozygous SNP locus.

II Detection Results of Disease-Resistant SNP Marker Based on S. chuatsiIL-6 Gene

The same population of S. chuatsi was bred under the same feedingconditions, and after breeding for about two months, 100 Mandarin fishwere randomly selected for challenge experiments from the culturedpopulation. Each fish was intraperitoneally injected with infectiousspleen and kidney necrosis virus (ISKNV) (also known as iridescentvirus), 10×TCID50 ISKNV. After observing for 10 days, it was observedthat the disease symptoms are the same as those of the ISKNV infectionin the natural environment. The diseased fish swam slowly on the watersurface, or evenly floated on the water surface, the body surface wasundamaged and the colour of body was white. The fish gills were ischemicwhitish. By means of anatomy, it was found that there were ascites inenterocoelia, the liver, the stomach wall and the intestinal wall werecongestive, and there was yellow effusion in intestinal tract. While thedisease-resistant fish were normal all the time. The PCR detectionindicated that the head kidney of the diseased S. chuatsi wereISKNV-positive, this shows that the death was caused by ISKNV infection.

9 undiseased fish and 3 diseased fish (marked as E05, H07, A07) wereselected, and a small quantity of tail fin was cut and placed inabsolute ethyl alcohol and stored at 4° C., respectively. In terms ofthe detection method of disease-resistant SNP marker as described inabove examples, the PCR amplification product was obtained, as shown inFIG. 3, after gel extraction, sequencing was performed and multiplesequence alignment was performed by DNAMAN, and the SNP locus associatedwith virus disease resistance was found at 233 bp of a nucleotidesequence fragment, as shown in FIG. 5.

The peak profile of suspected mutant base at 233 bp in the abovesequence was observed by Chromas software, as shown in FIG. 6, FIG. 7,and FIG. 8. At the corresponding bases G and A, the peaks are single andthere are no impurity peaks. From this, the base at 233 bp is mutatedfrom G to A, that is G233A. Because the mutation was in the ReverseComplement sequence of DNA, that is, a base sequenceAGCTCTTTTGCCGTCGACAAGGAA (SEQ ID NO: 16) (FIG. 5) adjacent to 233 bp ofa nucleotide sequence fragment, reverse complement to a base sequenceadjacent to 1733 bp of IL-6 gDNA sequence (SEQ ID NO: 2.), in theoriginal DNA sequence, SNP locus was at C1744T. The three samples withbase mutation (E05, H07, A07) (AGCTCTTTTGCCATCGACAAGGAA (SEQ ID NO: 17)(FIG. 5)) all were S. chuatsi susceptible to virus. Consequently, themutation from G to T was generated at 1744 bp of SNP of IL-6 gene of S.chuatsi susceptible to virus. The base at 1744 bp of S. chuatsisusceptible to virus is T, and the base at 1744 bp of antivirus S.chuatsi is C, the virus is infectious spleen and kidney necrosis virus(ISKNV). By means of this method, the antivirus S. chuatsi can bedistinguished from S. chuatsi susceptible to virus.

The above preferred embodiments are described for illustration only, andare not intended to limit the scope of the invention. It should beunderstood, for a person skilled in the art, that various improvementsor variations can be made therein without departing from the spirit andscope of the invention, and these improvements or variations should becovered within the protecting scope of the invention.

What is claimed is:
 1. A detection method for a disease-resistant SNPmarker of a Siniperca chuatsi IL-6 gene, comprising steps of: (1)designing a primer for amplifying a disease-resistant SNP locusaccording to an IL-6 gDNA sequence containing an intron of the Sinipercachuatsi IL-6 gene, and amplifying the Siniperca chuatsi IL-6 gene toobtain an amplification product; (2) sequencing the amplificationproduct and finding out SNPs loci relevant to virus disease resistance,and determining a SNP locus according to a DNA peak profile, wherein theSiniperca chuatsi IL-6 gene has a cDNA sequence shown in SEQ ID NO: 1.2. The detection method for a disease-resistant SNP marker as claimed inclaim 1, wherein in the step (1), the IL-6 gDNA sequence is obtained bydesigning and amplifying a specific primer for Siniperca chuatsi IL-6gene, and the IL-6 gDNA sequence having a nucleotide sequence shown inSEQ ID NO:
 2. 3. The detection method for a disease-resistant SNP markeras claimed in claim 2, wherein the specific primer is: (SEQ ID NO: 12)IL-6-1F: 5′-CTCAGCATCAGCGGAAACTC-3′; (SEQ ID NO: 13)IL-6-1R: 5′-TGCCCCTGTTGGCCATACTT-3′.


4. The detection method for a disease-resistant SNP marker as claimed inclaim 1, in the step (1), the primer for amplifying a disease-resistantSNP locus is: (SEQ ID NO: 14) IL-6- L1F: 5′-AACCCAAAGAGGCAGGTGAC-3′;(SEQ ID NO: 15) IL-6- L1R: 5′-ACCATCCAATTTCCCTGAGGT-3′.


5. The detection method for a disease-resistant SNP marker as claimed inclaim 1, wherein in the step (2), multiple sequence alignment isperformed on the sequencing results by DNAMAN software and the suspectedSNPs loci are found out, and DNA sequencing chromatogram is observed viaChromas software, single peak is a homozygotic SNP locus, and the nestedpeak is a heterozygous SNP locus.
 6. The detection method for adisease-resistant SNP marker as claimed in claim 1, wherein the SNPmarker is located at 1744 bp of the gDNA sequence of Siniperca chuatsihaving a nucleotide sequence shown in SEQ ID NO: 2, the base at 1744 bpof Siniperca chuatsi susceptible to virus is T, and the base at 1744 bpof antivirus Siniperca chuatsi is C, the virus is infectious spleen andkidney necrosis virus.